Introduction
There are a vast myriad of Escherichia coli (E. coli) species. Each strain is slightly different from the next. Many strains are an active part of our everyday, healthy lives. However, some strains can cause serious illness and even death. In order to help prevent health risks posed by E. coli, it would be helpful to understand the genetic differences between the helpful and harmful strains. It is known that these strains often differ in the content of their genomes. One key point of difference occurs in the distribution of mobile genetic elements—the insertion of sequences, transposons, and bacterial viruses (integrated as prophages) (Christie).

Prophages give their bacterial hosts a selective advantage by providing genes that encode for toxins, virulence factors, and/or other functions (Christie). Several homologs to phage DNA have already been connected to the pathogenicity of E. coli in poultry (Stocki). Several people in Washington died after eating at Jack in the Box in January of 1993 because the meat contained E. coli that produced shiga toxin (encoded by the stx gene). The stx gene comes from prophages of the lambdoid family (Wagner).

P2 is a double-stranded DNA virus with a known genomic sequence. Furthermore, P2 phages are relatively widespread among the gamma Proteobacteria. P2 goes through two different life cycles: lytic and lysogenic. In the lytic stage, P2 replicates itself until the host cell lyses (bursts). On the other hand, during the lysogenic stage, the phage’s DNA is integrated into the genome of the host. Consequently, most P2 genes are dormant until the phage enters the lytic cycle. However, some phage genes may be expressed and have subtle effects on the phenotype of the host organism. The process by which a phage in the lysogenic state controls the phenotype of its host is called lysogenic conversion (UIUC.edu). Does a certain pathogenic E. coli contain P2-related prophages in its genome (in whole or in part)? If so, what genes are present? Do these genes confer any phenotypic difference? Are these the genes that contribute to the virulence of pathogenic E. coli? Do these bacteria contain a P2 lysogenic conversion gene? (Christie)


Methods
PCR (polymerase chain reaction) primers must be made to screen a given E. coli for P2-like prophages. To construct the necessary primers, known E. coli genomes must be collected and aligned. These genomes must contain similar or conserved sequences from the P2 prophage genome—preferably about seventeen to twenty-four base pairs long. This search will begin with the P gene, the P2 virus’s capsid protein gene. If unsuccessful, the nearby Q, O, N, M, and L genes will be compared. The genetic sequence will be taken from E. coli and Salmonella genomes in the NCBI database. Only coding regions of DNA will be considered.

Once primers have been created, PCR will be run on various strains of E. coli. This will amplify (by making numerous clones of) the sections of DNA that match those between the two primers. Strains from Gail Christie’s collection will be tested and used as positive and negative controls. Additionally, strains of diarrheagenic E. coli from Iruka Okeke’s laboratory at Haverford College will be tested. Strains with positive PCR results are candidates for containing P2-related prophages.
A Southern Blot will be run on the strains with a positive PCR. The entire P2 genome will be used to probe the DNA. This will help to elucidate which cloned fragments are present in the E. coli strain and which are not present. The DNA of the E. coli strains will be digested using a restriction nuclease. Then the DNA will be separated by size using gel electrophoresis. The fractionated DNA molecules will be transferred to a sheet of nitro-cellulose paper. This paper is incubated in a solution containing a labeled DNA probe (P2). E. coli strands that are complementary to the P2 probe will be identified.
If time permits and P2 genes are found, a lysogenic conversion gene will be sought after. If identified, targeted gene knockout (a known process for altering E. coli) will be performed. Then, the E. coli will be observed to see if any lysogenic conversion takes place.


Possible Results and their Implications
PCR is a very sensitive procedure. Accordingly, it can locate a single molecule of DNA in a sample and duplicate the sequence that is in between the two primers. However, the process is also very specific. PCR will fail to recognize similar sequences. Since we are dealing with conserved sequences, they will be similar but not necessarily identical. This may result in some strains yielding a negative result when they actually contain a P2-like prophage. Furthermore, our primers will be biased because it will be based upon the P2-like prophages of E. coli and salmonella strains that have previously been sequenced (we cannot account for variations that exist in P2-like prophages that have not been sequenced). Errors aside, a positive PCR result means that the strain being tested matches the primer and the DNA will be repeatedly cloned. A negative result will occur when the primer does not complement the DNA. Consequently, the DNA will not be duplicated. If all the strains yield negative results, a new primer must be tested.

Southern blotting is slightly different. It is less specific but will give more detailed results. Individual genes can be located and isolated. Single nucleotide differences will be ignored, but Southern blotting will determine which P2 genes are present in the E. coli strain being tested.

The next step, after this data is acquired, would be to target the lysogenic conversion genes and/or the virulence genes. This could provide insight into how to cure or help prevent the diseases caused by pathogenic E. coli that contain P2-like prophages.

References

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter; Molecular Biology of the Cell, Fourth Edition; Garland Science: New York, 2002; ch. 8.

Christie, Gail E; http://www.vcu.edu/csbc/bbsi/people/faculty/gail_christie.html accessed 6/2003

Labrie, Steve; Moineau, Sylvain; “Multiplex PCR for Detection and Identification of Lactococcal Bacteriophages” Applied and Environmental Microbiology; March 2000; vol. 66, no. 3, p987-94.

Mirold, Susanne; Rabsch, Wolfgang; Tschape, Helmut; Hardt, Wolf-Dietrich; “Transfer of the Salmonella Type III Effector sopE between Unrelated Phage Families” Journal of Molecular Biology; 2001; vol. 312, p7-16.

Nataro, James P; Kaper, James B; “Diarrheagenic Escherichia coli” Clinical Microbiology Reviews; January 1998; vol. 11, no. 1, p142-201.

Okeke, Iruka N; http://www.haverford.edu/biology/Okeke/Research.htm accessed 6/2003

Stocki, Stacy L; Babiuk, Lorne A; Rawlyk, Neil A; Potter, Andrew A; Allan, Brenda J; “Identifcation of genomic differences between Escherichia coli strains pathogenic for poultry and E. coli K-12 MG1655 using suppression subtractive hybridization analysis”; Microbial Pathogenesis; 2002; vol. 33, p289-98.

Strauch, Eckhard; Lurz, Rudi; Beutin, Lothar; “Characterization of a Shiga Toxin-Encoding Temperate Bacteriophage of Shigella sonnei” Infection and Immunity; December 2001; vol. 69, no. 12, p7588-95.

Wagner, Patrick L; Neely, Melody N; Zhang, Xiaoping; Acheson, David WK; Waldor, Matthew K; Friedman, David I; “Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coli Strain” Journal of Bacteriology; March 2001; vol. 183, No. 6, p2081-5

Vallance, BA; Finlay, BB. “Exploitation of host cells by enteropathogenic Escherichia coli” Proceedings of the National Academy of the United States of America. August 1, 2001. vol. 97, no. 16, p8799-8806.

http://www.life.uiuc.edu/micro/316/topics/phage/phage-virulence.html accessed 7/2003